The goals of the proposed research are to advance our knowledge about how oncogenic transformation perturbs cell differentiation programs and to understand how these perturbations can be reversed to reestablish differentiation and terminal cell divisions in tumor cells. It is now clear that tumor cells can, under appropriate conditions, reinitiate and complete their normal differentiation program. Our approach is to investigate the molecular basis for the block to differentiation present in murine erythroleukemia (MEL) cell lines and the events occurring when the cells are induced to reenter their terminal differentiation program. Recent work in our laboratory indicates that deregulated expression of an Ets family transcription factor, PU.1 (Spi-1), is principally responsible for the block to differentiation present in MEL cells. We purpose to determine the molecular mechanisms by which PU.1 blocks erythroid differentiation. One approach will be to investigate which transcription factor functions of PU.1 are required for blocking MEL cell differentiation and to identify the downstream gene targets that are regulated by PU.1. A parallel approach will be to investigate the possibility that PU.1 blocks differentiation by directly interacting with proteins that are required for red blood cell differentiation. Other studies in our laboratory indicate that changes in expression of cyclin-dependent kinases and their inhibitors may be involved both in the decision of the cells to differentiate and in implementing the final cell divisions. We propose to carry out transfection experiments to determine whether changes in these cell cycle regulators contribute to controlling these two aspects of the differentiation program. Understanding the events occurring in tumor cells when they are forced back into terminal differentiation will contribute to our knowledge about the control of cell proliferation and differentiation as well as provide opportunities for development of therapies based on the capacity of tumor cells to resume terminal cell divisions.